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Mutation analysis, heterologous expression, and characterization of human glucocerebrosidase

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dc.contributor.author Sinclair, Graham Bernard
dc.date.accessioned 2018-09-21T21:46:56Z
dc.date.available 2018-09-21T21:46:56Z
dc.date.copyright 2001 en_US
dc.date.issued 2018-09-21
dc.identifier.uri https://dspace.library.uvic.ca//handle/1828/10086
dc.description.abstract Gaucher disease, the most common lysosomal storage disorder, results from a deficiency in the enzyme glucocerebrosidase. Inherited as an autosomal recessive disorder, Gaucher disease is clinically heterogeneous with both non-neuronopathic (Type 1) and neuronopathic (Types 2 and 3) subtypes. Although over 100 mutations in the glucocerebrosidase (GBA) gene have been identified, there still exists a poor correlation between individual genotypes and observed phenotypes, particularly for the neuronopathic subtypes. Using DNA isolated from archival tissue samples and standard molecular biology techniques, two novel and two rare mutations were identified in three individuals with neuronopathic Gaucher disease. One mutation identified only in aboriginals of Cree descent was further characterized by heterologous expression in baculovirus-infected Sf9 cells and displayed moderate levels of residual enzyme activity, despite the corresponding disease severity observed. Heterologous expression studies were extended to examine systems for high-level glucocerebrosidase expression for biochemical analysis and biotherapeutics. While the methylotrophic yeast Pichia pastoris was found to express minimal amounts of human glucocerebrosidase even when selected for high gene copy number, stable transfected Sf9 cells were found to produce functional glucocerebrosidase at a level of 1.0–1.3mg/L of cell culture. A subsequent analysis of synonymous codon usage bias in Pichia pastoris identified a significant difference in codon choice between the expression host and the GBA gene. Codon optimization studies using a 5′ fragment of the GBA gene fused to a luciferase reporter gene found that alterations in both G+C content and codon bias increased expression levels 7.5 to 10 fold. This suggests that codon optimization of the entire GBA gene could significantly improve production levels of this important enzyme in Pichia pastoris and other expression hosts. en_US
dc.language English eng
dc.language.iso en en_US
dc.rights Available to the World Wide Web en_US
dc.subject Gaucher's disease en_US
dc.subject Genetic aspects en_US
dc.subject Lysosomes en_US
dc.title Mutation analysis, heterologous expression, and characterization of human glucocerebrosidase en_US
dc.type Thesis en_US
dc.contributor.supervisor Choy, Francis Y. M.
dc.degree.department Department of Biology en_US
dc.degree.level Doctor of Philosophy Ph.D. en_US
dc.description.scholarlevel Graduate en_US


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